Biomedical subjects
Publications and source records attributed to E Norrby.
These results demonstrated that cross-reactions between mumps virus, PIV-2, PIV-4, and SV5 involve both internal components and surface glycoproteins and that these viruses form a separate group within the paramyxovirus genus.
Measles virus (MV) proteins were efficiently expressed in COS and Vero cells from vectors based on the strong cytomegalovirus enhancer-promoter and the simian virus 40 origin of replication. When expressed alone, nucleocapsid protein (N) migrates predominantly into the nucleus whereas phosphoprotein (P) is located in the cytoplasm. Coexpression of N and P proteins results in retention of the N protein in the cytoplasm, as seen also in infected cells. The retention of N protein is due to specific interactions with the P protein since coexpression of N with either the matrix or the hemagglutinin protein had no effect. Mapping of the regions of N-P interactions on P protein revealed that the carboxy-terminal 40% of P was sufficient for specific binding to N; however, the carboxy-terminal 60% of P was required for retention of N in the cytoplasm. Thus, the V and C proteins encoded within the first half of the P gene are not involved in the cytoplasmic retention of N protein. N protein might be fortuitously targeted to the nucleus as a result of its many basic amino acids, presumably destined to interact with the MV genome. However, this set of experiments has allowed to analyze in vivo the interactions between the N and P proteins.
The role of individual amino acids in binding human and macaque antibodies were determined in the human immunodeficiency virus type 1 (HIV-1) gp41, residues 594-613, and for human antibodies in the hepatitis B (HB) virus core/e antigens (HBc/eAg), residues 121-140. Decapeptides with 9 amino acids (aa) overlap were synthesised using a rapid method for simultaneous multiple peptide synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) protection for the alpha-amino group of the aas. One coupling cycle including washing steps was performed within 60-90 min. The crude products were analysed by reversed-phase HPLC and PD-mass spectrometry. With the 11 decapeptides covering residues 594-613 of HIV-1 gp41, the sequences SGKLI at aa 599-603 was found to be the main recognition site for 19 human anti-HIV positive sera. Two macaques repeatedly immunized with a peptide covering aa 594-613 of gp41, preferentially recognised the sequence CTTAVPW at residues 604-610 after 1-2 months of immunisation. One macaque also recognised the sequence CSGKLI, with sera sampled greater than 10 months after start of immunisation. Out of 9 human sera from patients with chronic HB, and reactive to a peptide covering residues 121-140 of HBc/eAg, 8 were found to recognise the sequence TPPA at residues 128-131, with an individual variation within residues 125-133 in regard to N- and C-terminal ends of the recognised antigenic site. Thus, human recognition of this antigenic site overlaps the reported T- and the B-cell recognition site found in mice. We believe that this simple and rapid approach to obtain large numbers of immunologically active peptides can be useful for most laboratories interested in the immunological characterisation of proteins.
Lymphocytic interferon gamma (IFN-gamma) production and major histocompatibility complex (MHC) antigen induction were studied in experimental measles and vesicular stomatitis virus infections in the brain. Fifteen-day-old Sprague-Dawley rats injected intracerebrally with the HNT strain of measles virus showed already within 1 day after infection an increased number of cells producing IFN-gamma in the spleen, cervical lymph nodes and leptomeninges. These rats recovered after a transient neuronal infection in the brain. Rats infected intracerebrally with vesicular stomatitis virus, on the other hand, all succumbed after 2 days and showed no IFN-gamma production in lymphoid cells. Immunohistochemically MHC class I antigen appeared in infected and uninfected cells in the brain during replication of both viruses. A role for the recently discovered nerve fibres with IFN-gamma-like immunoreactivity, which are normally present in the brain, in the MHC antigen induction is discussed.
The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.
A rapid and simple purification procedure for human immunodeficiency virus type 1 (HIV-1) proteinase from a synthetic gene expressed in Escherichia coli has been developed. The synthetic gene was constructed from oligonucleotides containing several restriction enzyme sites in order to allow simple construction of homologous genes. The protein was translated as a precursor which was autocatalytically processed into the mature protein as shown by N-terminal sequence analysis of the purified protein. Immunoblot analysis was used to verify the nature of the expression product and it was found that 2 of 10 anti-peptide antibodies, covering the whole proteinase sequence, were able to react with the enzyme in crude bacterial lysates. These two anti-peptide antibodies represent a continuous sequence partially overlapping the active site. The purification involves two initial precipitation steps followed by cation-exchange and size-exclusion chromatography. A high yield and a high specific activity were achieved.
The aim of this study was to determine if protection against an infectious human immunodeficiency virus type 2 (HIV-2) challenge could be obtained in cynomolgus macaques by active immunization using whole killed virus vaccine. Four monkeys were immunized with killed HIV-2SBL-6669, two of them with five intramuscular (im) injections of viral preparation containing 100 or 300 micrograms protein emulsified in incomplete Freund's adjuvant (IFA) and the two remaining received four im injections of 25-50 micrograms viral protein in iscoms. Each of the four vaccinated cynomolgus monkeys, along with four unvaccinated controls, were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses (ID50) of live HIV-2SBL-6669. All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins before challenge exposure to HIV-2, but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The two monkeys immunized with killed virus in IFA have shown no evidence of infection nine months after challenge with live virus. When blood and lymph node cells from these animals were transfused into naive cynomolgus monkeys, the recipients remained free of infection. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with iscom-associated viral antigens, which had a low content of envelope gp125 antigen. The demonstration of vaccine-induced protection against HIV-2 in a nonhuman primate raises hope for effective immunization against HIV infections in humans as well.
The occurrence of dominant linear antigenic sites in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2) was evaluated. Twenty-five peptides representing different regions of HIV-2, strain SBL-6669, were synthesized. For comparison the corresponding peptides of HIV-1, strain BRU, were also prepared. The peptides were tested in enzyme-linked immunosorbent assay (ELISA) with human sera from individuals with proven HIV-1 or HIV-2 infection and simian sera from animals infected with HIV-2 or simian immunodeficiency virus of sooty mangabay monkey origin (SIVsm). Four major antigenic regions were identified. Peptides representing parts or the whole V3 (neutralizing loop) region and an additional stretch of amino acids located at the carboxy terminal of this region showed considerable reactivity. This reaction was predominantly type specific, but some heterotypic reactivity was also seen. Peptides representing the carboxy terminal 21 amino acids of the V3 region of the type-related viruses HIV-2 and SIVsm allowed selective identification of strain-specific antibodies. A second major antigenic region was found close to the carboxy terminal end of the large glycoproteins. This region was cross-reactive between the two types. The two additional dominating antigenic regions were located in the amino terminal region of the transmembrane glycoprotein. One region has previously been shown to be a uniquely antigenic type-specific site. The other region was also type-specific, but was identified only in HIV-2, amino acids Glu634-Lys649. Excellent facilities are available for the design of not only type-unique site-specific serological tests but potentially also type-cross-reactive and strain-specific assays.
Mumps virus caused a partially lytic infection in cultivated rat embryonic dorsal root ganglion neurons with a restricted formation of viral components. The neuronal degeneration was markedly enhanced by increasing the calcium concentration of the medium and could be almost totally inhibited by the dihydropyridine calcium channel antagonist nifedipine. The drug had no effect on a productive and completely lytic Sendai virus infection of the neurons. A previous neurophysiological study has shown that a reduced calcium influx occurs during the action potential early during the infection. The present study suggests a crucial role of calcium in neurodegeneration induced by certain viruses.
The possibility of using simian immunodeficiency virus (SIV)-infected macaques to study pathogenic events linked to HIV infection of the brain prompted us to investigate some of the virological features in SIV-infected macaques. Nine cynomolgus macaques were inoculated with SIVsm and killed at different times. We successfully isolated virus from the blood of all the animals and from the brains of eight. These results point to the early and regular spread of this lentivirus to the brain. Neutralizing activity was studied in the serum and cerebrospinal fluid specimens obtained from these macaques against a selected group of isolates. Cerebrospinal fluid did not show any neutralizing activity. Our findings integrate the observations from HIV-1 infection in man and indicate that SIV infection of macaques is a useful model for studying pathogenic events of brain infection.
A library of cDNA clones from mRNA of human parainfluenza type 2 virus (PIV2) was constructed and the nucleotide sequence of the fusion (F) glycoprotein gene determined. The F gene boundaries were obtained by primer extension sequencing on F mRNA and on viral genomic RNA. The mRNA coding for the F glycoprotein is composed of 1918 nucleotides. It contains a single large open reading frame that encodes a protein of 551 amino acids with an Mr of 59586. The predicted PIV2 F protein contains the cleavage-activation site (amino acids 101 to 106) including two Arg and two Lys residues, where the protein is cleaved by host protease into F1 and F2 subunits by analogy with other paramyxoviruses. Three hydrophobic domains are recognized, the signal peptide (amino acids 1 to 21), that is cleaved off in the mature protein, the fusion peptide (amino acids 107 to 132) at the cleavage-generated N terminus of subunit F1 and the membrane anchorage region (amino acids 486 to 513) near the C terminus of the protein. The predicted F protein has six potential glycosylation sites and 10 of the 12 Cys residues present have conserved positions as compared with those of other paramyxovirus F proteins. Primer extension sequencing on viral RNA gave the 3' end sequence as UAAAUUCU6 followed by the 5' end of the haemagglutinin-neuraminidase (HN) gene thus establishing the order of genes coding for the viral glycoproteins of PIV2 as 5'-F-HN-3'.
The amino acid composition of the two surface proteins of the recently isolated morbillivirus phocid distemper virus (PDV) were deduced from the nucleotide sequence. The fusion (F) protein of PDV exhibited characteristics similar to those of other morbillivirus F proteins. The overall amino acid similarity with its closest homologue, canine distemper virus (CDV), was 72%. From the context of the starting codons and the requirement for a hydrophobic signal peptide, it is likely that translation of the PDV F mRNA starts at the third AUG, corresponding to codon 95 in the long open reading frame of the PDV F gene. After removal of the signal peptide, F0 starts at amino acid 105. From this position the F protein of PDV and CDV exhibit 84% amino acid similarity. The PDV haemagglutinin (H) protein showed 74% amino acid similarity with CDV H protein and highly conserved features responsible for the tertiary structure. Despite these similarities, the two H proteins show marked antigenic differences when probed with monoclonal antibodies. Earlier studies have indicated that rinderpest virus (RPV) is the prototype virus of the morbillivirus genus, from which first CDV/PDV and later measles virus (MV) evolved. From the close relationship shown in this study, it is likely that the divergence of CDV and PDV occurred after MV evolved from RPV.
Respiratory syncytial virus strains isolated from infants and children hospitalized with acute respiratory tract disease in Huntington, W. Va., from July 1979 through June 1988 were categorized by subgroup by using two panels of monoclonal antibodies specific for subgroup A and subgroup B. Of 405 strains tested, 319 (78.8%) were subgroup A, 73 (18.0%) were subgroup B1, and 13 (3.2%) were subgroup B2. The subgroup B2 strains occurred as a single-season epidemic during the epidemiologic year July 1980 to June 1981, except for one strain isolated in the epidemiologic year 1978 to 1979. Subgroup B strains were associated with significantly fewer bronchiolitis illnesses than subgroup A strains. The severity of illnesses caused by subgroup B1 and B2 strains was the same.
A fluorescence antibody test with monoclonal antibodies was used to determine the subgroup (A or B) of respiratory syncytial virus from infants hospitalized in Caen, France, over eight consecutive epidemics from 1982 to 1990. From 1982 to 1985, 27 (30%) out of 90 frozen nasal slides were classified as subgroup A strains and 63 (70%) were classified as subgroup B. B strains predominated over A in 1983-1984 and 1984-1985. From 1985 to 1990, 284 respiratory syncytial virus field strains were reisolated from frozen materials; 115 (40.5%) were typed as subgroup A and 169 (59.5%) were typed as subgroup B. In 1985-1986, 1986-1987, and 1988-1989, both subgroups were present in almost equal numbers; subgroup A (88.3%) predominated in 1987-1988, and subgroup B (84.5%) predominated in 1989-1990. In conclusion, both subgroups occur together each year, and one subgroup rarely predominates, e.g., subgroup A in 1987-1988 and subgroup B in 1983-1984 and 1989-1990. Therefore, there is a gradual change of the predominant subgroup into another over a period of about 5 years; the relative frequency of subgroup A strains increased from 1983 to 1988, whereas the percentage of subgroup B decreased during the same period.
One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.
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The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.
An acute infection with simian immunodeficiency virus (SIVSM) in cynomolgus monkeys was used to evaluate the antiviral effects of 3'-fluorothymidine (FLT) and 3'-azidothymidine [zidovudine (ZDV)]. Neither compound prevented the infection despite dosing prior to virus inoculation. FLT was about ten times more potent than ZDV in delaying the appearance of SIVSM antigen in the monkeys. The serum half-life of FLT was longer than that of ZDV and ZDV was bound to plasma proteins to about 60% while FLT was virtually unbound. It is proposed that the in vivo difference in potency between ZDV and FLT could, at least partly, be explained as the combined effects of a longer plasma half-life and a higher free concentration of FLT and possibly a higher intracellular concentration of the triphosphate of FLT.